2,3,7,8-Tetrachlorodibenzo-p-dioxin has both pro-carcinogenic and anti-carcinogenic effects on neuroendocrine prostate carcinoma formation in TRAMP mice.

It is well established that the prototypical aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can both cause and protect against carcinogenesis in non-transgenic rodents. But because these animals almost never develop prostate cancer with old age or after carcinogen exposure, whether AHR activation can affect cancer of the prostate remained unknown. We used animals designed to develop this disease, Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, to investigate the potential role of AHR signaling in prostate cancer development. We previously reported that AHR itself has prostate tumor suppressive functions in TRAMP mice; i.e., TRAMP mice in which Ahr was knocked out developed neuroendocrine prostate carcinomas (NEPC) with much greater frequency than did those with both Ahr alleles. In the present study we investigated effects of AHR activation by three different xenobiotics. In utero and lactational TCDD exposure significantly increased NEPC tumor incidence in TRAMP males, while chronic TCDD treatment in adulthood had the opposite effect, a significant reduction in NEPC incidence. Chronic treatment of adult TRAMP mice with the low-toxicity selective AHR modulators indole-3-carbinol or 3,3'-diindolylmethane did not significantly protect against these tumors. Thus, we demonstrate, for the first time, that ligand-dependent activation of the AHR can alter prostate cancer incidence. The nature of the responses depended on the timing of AHR activation and ligand structures.

Intersection of AHR and Wnt signaling in development, health, and disease.

The AHR (aryl hydrocarbon receptor) and Wnt (wingless-related MMTV integration site) signaling pathways have been conserved throughout evolution. Appropriately regulated signaling through each pathway is necessary for normal development and health, while dysregulation can lead to developmental defects and disease. Though both pathways have been vigorously studied, there is relatively little research exploring the possibility of crosstalk between these pathways. In this review, we provide a brief background on (1) the roles of both AHR and Wnt signaling in development and disease, and (2) the molecular mechanisms that characterize activation of each pathway. We also discuss the need for careful and complete experimental evaluation of each pathway and describe existing research that explores the intersection of AHR and Wnt signaling. Lastly, to illustrate in detail the intersection of AHR and Wnt signaling, we summarize our recent findings which show that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced disruption of Wnt signaling impairs fetal prostate development.

In utero exposure to TCDD alters Wnt signaling during mouse prostate development: linking ventral prostate agenesis to downregulated ß-catenin signaling.

In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes ventral prostate agenesis in C57BL/6J mice by preventing ventral prostatic budding in the embryonic urogenital sinus (UGS). TCDD (5 µg/kg, po) administered to pregnant dams on embryonic day 15.5 (E15.5) activates the aryl hydrocarbon receptor in the UGS mesenchyme, disrupting the mesenchymally derived paracrine signaling that instructs epithelial prostatic budding. How TCDD alters the mesenchymal milieu is not well understood. We previously showed that TCDD disrupts some aspects of Wnt signaling in UGSs grown in vitro. Here we provide the first comprehensive, in vivo characterization of Wnt signaling in male E16.5 UGSs during normal development, and after in utero TCDD exposure. Vehicle- and TCDD-exposed UGSs were probed by in situ hybridization to assess relative abundance and localization of RNA from 46 genes that regulate Wnt signaling. TCDD altered the staining pattern of five genes, increasing staining for Wnt10a and Wnt16 and decreasing staining for Ror2, Rspo2, and Wif1. We also used immunohistochemistry to show, for the first time, activation of ß-catenin (CTNNB1) signaling in ventral basal epithelium of control UGSs at E16.5. This onset of CTNNB1 signaling occurred immediately prior to the initiation of ventral prostatic budding and is characterized by a pronounced increase in CTNNB1 nuclear localization and subsequent expression of the CTNNB1 signaling target gene, Lef1. In utero TCDD exposure prevented the onset of CTNNB1 signaling and LEF1 expression in the ventral basal epithelium, thereby elucidating a likely mechanism by which TCDD contributes to failed prostatic budding in the ventral UGS.

TCDD inhibition of canonical Wnt signaling disrupts prostatic bud formation in mouse urogenital sinus.

In mice, in utero exposure to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) reduces the number of dorsolateral prostatic buds resulting in a smaller dorsolateral prostate and prevents formation of ventral buds culminating in ventral prostate agenesis. The genes and signaling pathways affected by TCDD that are responsible for disrupting prostate development are largely unknown. Here we show that treatment of urogenital sinus (UGS) organ cultures with known inhibitors of canonical Wnt signaling also inhibits prostatic bud formation. In support of the hypothesis that TCDD decreases canonical Wnt signaling, we identify inhibitory effects of TCDD on multiple components of the canonical Wnt signaling pathway in the UGS that temporally coincide with the inhibitory effect of TCDD on prostatic bud formation: (1) expression of R-spondins (Rspo2 and Rspo3) that promote canonical Wnt signaling is reduced; (2) expression of Lef1, Tcf1, and Wif1, established canonical Wnt target genes, is decreased; (3) expression of Lgr5, a RSPO receptor that activates canonical Wnt signaling, is reduced; and (4) expression of Dickkopfs (Dkks), inhibitors of canonical Wnt signaling, is not increased by TCDD. Thus, the TCDD-induced reduction in canonical Wnt signaling is associated with a decrease in activators (Rspo2 and Rspo3) rather than an increase in inhibitors (Dkk1 and Dkk2) of the pathway. This study focuses on determining whether treatment of TCDD-exposed UGS organ cultures with RSPO2 and/or RSPO3 is capable of rescuing the inhibitory effects of TCDD on canonical Wnt signaling and prostatic bud formation. We discovered that each RSPO alone or in combination partially rescues TCDD inhibition of both canonical Wnt signaling and prostatic bud formation.

Wnt inhibitory factor 1 (Wif1) is regulated by androgens and enhances androgen-dependent prostate development.

Fetal prostate development from urogenital sinus (UGS) epithelium requires androgen receptor (AR) activation in UGS mesenchyme (UGM). Despite growing awareness of sexually dimorphic gene expression in the UGS, we are still limited in our knowledge of androgen-responsive genes in UGM that initiate prostate ductal development. We found that WNT inhibitory factor 1 (Wif1) mRNA is more abundant in male vs. female mouse UGM in which its expression temporally and spatially overlaps androgen-responsive steroid 5α-reductase 2 (Srd5a2). Wif1 mRNA is also present in prostatic buds during their elongation and branching morphogenesis. Androgens are necessary and sufficient for Wif1 expression in mouse UGS explant mesenchyme, and testicular androgens remain necessary for normal Wif1 expression in adult mouse prostate stroma. WIF1 contributes functionally to prostatic bud formation. In the presence of androgens, exogenous WIF1 protein increases prostatic bud number and UGS basal epithelial cell proliferation without noticeably altering the pattern of WNT/ß-catenin-responsive Axin2 or lymphoid enhancer binding factor 1 (Lef1) mRNA. Wif1 mutant male UGSs exhibit increased (Sfrp)2 and (Sfrp)3 expression and form the same number of prostatic buds as the wild-type control males. Collectively our results reveal Wif1 as one of the few known androgen-responsive genes in the fetal mouse UGM and support the hypothesis that androgen-dependent Wif1 expression is linked to the mechanism of androgen-induced prostatic bud formation.

Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis.

Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGFbeta-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis.

Characterization of the six zebrafish clade B fibrillar procollagen genes, with evidence for evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide.

Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I-III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-alpha1(V), pro-alpha3(V), pro-alpha1(XI) and pro-alpha2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-alpha1(V), pro-alpha3(V)a and b, pro-alpha1(XI)a and b, and pro-alpha2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH(2)-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system.

Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4.

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-beta-like bone morphogenetic proteins (BMPs) 2-7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2-7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2-7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of approximately 11 nM, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.